Circadian clocks, described in organisms ranging in complexity from unicells to mammals, functions to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology, including endocrine function, sleep/wake cycles, psychiatric illness, as well as drug tolerance and effectiveness. Additionally, cell division in many human tissues is clock- regulated, providing the basis for promising new approaches to cancer chemotherapy. Our long term goal is to understand the molecular and biochemical basis for circadian rhythmicity. The clock in all organisms is assembled within the cell and clock mechanisms are evolutionarily conserved; thus, simple eukaryotes provide appropriate experimental systems to investigate the clock and efficiently achieve these goals. Our aims will be carried our in Neurospora crassa, a well-defined model organism with one of the most highly characterized clocks. An important aspect of circadian rhythmicity is clock-control of gene expression. However, little is known about how this regulation takes place or of the oscillator components that signal time information in the cell. Our preliminary results in Neurospora lead us to hypothesize that there are distinct rhythmic output pathways which are driven by one or more oscillators. To test this hypothesize, we are focusing our studies on identifying and describing circadian output pathways, and investigating the regulation of clock-controlled genes. In Specific Aim 1 we will use a genetic mutant screen to identify genes involved in the regulation of circadian output based on an automated rhythmic luciferase reporter system. Fusion of clock regulatory sequences to luciferase will permit the isolation of mutant strains that are altered or are deficient in their ability to relay temporal information. In Specific Aim 2 we will use transcriptional profiling to identify rhythmically expressed genes in Neurospora, to determine the prevalence of rhythmic gene expression, and to better understand the role of the clock in cell function. In Specific Aim 3 we will identify proteins that interact with the known clock component FRQ by screening for a) mutations that completely abolish rhythmicity is evident, and b) extragenic suppressors under growth conditions in which the strain is arrythmic. Together, the proposed experiments will permit a detailed understanding of how the clock functions within the cell.